With the advent of recombinant DNA technology, the use of microorganisms as mini-factories for the production of many useful proteins, enzymes, and other substances has become a routine occurrence. One broad group of microorganisms of choice for use as mini-factories has included organisms from the genus Escherichia and species coli commonly referred to as E. coli. Within this group is a widely used subgroup of organisms which are members of a strain referred to as the E. coli K-12 strain see "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology", (1987) Neidhardt, F. C. et al. (eds.), American Society for Microbiology, volume 2, chapter 72!.
Individual clones within the E. coli K-12 strain are particularly attractive host choices for recombinant DNA (rDNA) manipulations and heterologous protein production due to the many years of research on this strain. This research has established a fairly solid understanding of the genetic, biochemical and physiological characteristics of this strain. Moreover, E. coli K-12 strains support the replication of a large number of bacteriophage and plasmids that are potentially useful vectors in heterologous protein production. The intimate knowledge of this strain of bacterium and its compatible vectors have assured its preeminence in recombinant DNA developments. Furthermore, the E. coli K-12 strain has been shown to be ineffective in colonizing the human gut or in persisting in any environments outside of laboratory or industrial cultivation, an environmental safety characteristic having both commercial and regulatory significance.
Although the E. coli K-12 strain is uniquely different from other E. coli strains, it is, in fact, not a single entity. Rather, it is a family of related bacterial clones, all derived by genetic mutation from an original isolate see "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology" (1987)!. The E. coli K-12 strains used for both research and commercial purposes today are derivatives of mutant clones which were created and isolated in the first studies of this strain, using irradiation with X-rays, and later with UV and other chemical treatments to induce random mutations. Some of the genetic mutants or derivatives have evolved through purposeful selection and, thus, have well characterized mutations. It is, however, also recognized that many of the present day derivatives contain undetected and/or, as yet, uncharacterized allelic differences. Thus, present day members of the E. coli K-12 strain differ from one another by mutations, both spontaneous and induced, in one or many genes.
The presence of both known and unknown phenotypic characteristics and of known and unknown mutations in the genomes of E. coli K-12 strains, however, has deterred neither the use of these microorganisms as host cells for heterologous protein production nor the quest to improve the host cell quality of these microorganisms. For although E. coli K-12 strains are currently employed to produce such heterologous proteins as human insulin, growth hormone and the like, the search continues for ways to improve the production of such proteins in these microorganisms. It is of interest to researchers and commercial manufacturers alike to seek improvements in, for example, the overall amount of heterologous protein produced, or reductions in production costs, or increases in the absolute yield of heterologous protein produced in such organisms as E. coli K-12.
The novel strains of the present invention and novel methods set forth herein for their use provide for increases in both the amount (level) of heterologous protein produced by E. coli K-12 strains and for increases in the yield (amount which can be purified) of heterologous protein. The novel strains and methods of the present invention also provide for production cost savings.
In one aspect, the present invention relates to the correction of a previously known frameshift mutation present in many E. coli K-12 strains. Quite surprisingly, correction of this frameshift mutation will result in an increase in the amount and yield of heterologous protein produced in organisms with the corrected mutation and, will furthermore, provide production cost savings.
Specifically, it is known that many E. coli K-12 strains have a frameshift mutation in the rph gene (see Jensen, K. F., (1993), J. Bacteriol. 175:3401-3407; Womack, J. E. and O'Donovan, G. A. (1978), J. Bacteriol. 136:825-827; Machida, H. et al. (1970) Agr. Biol. Chem. 34:1129-1135; and Machida, H. and Kuninaka, A. (1969), Agr. Biol. Chem. 33:868-875. This frameshift mutation has been demonstrated to affect expression of the downstream pyrE gene resulting in depressed production of the pyrE gene product, phosphoribosyltransferase (ORPTase). Since ORPTase is responsible for the conversion of orotic acid to ortidine monophosphate, depressed levels of ORPTase result in an accumulation of orotic acid in the cell and growth medium during cell growth. Additionally, since ORPTase is an important pyrimidine pathway enzyme, it has been common practice to add uridine, when minimal growth media is used, to avoid possible pyrimidine starvation.
Recent studies by Jensen, K. F. (1993), however, have shown that the addition of uridine to the growth medium of E. coli K-12 strains with the rph frameshift mutation has no effect on the overall growth rate (doubling time) of these strains. Jensen, K. F. (1993) has also demonstrated that correction of the rph frameshift mutation in E. coli K-12 strains by genetic complementation with fully functional, but genetically unrelated rph and pyrE genes, has no affect on the growth rate (doubling time) of said organisms see Jensen, K. F., (1993)!. Thus, Jensen teaches that the presence of a rph frameshift mutation is not a growth-limiting defect in E. coli K-12 strains. The work of Jensen furthermore strongly suggests that there is no discernable gross metabolic benefit to be obtained by curing or correcting the rph frameshift mutation. Indeed, the work by Jensen seems to specifically demonstrate that the rph frameshift mutation does not create or produce any detectable harmful effects on the growth and reproduction of E. coli K-12 strains containing this frameshift mutation. Additionally, the accumulation of orotic acid, noted in E. coli K-12 strains containing the frameshift mutation, has been shown merely to be an indicator of the presence of the frameshift mutation. Neither Jensen nor others have reported any untoward effects of orotic acid over-production.
It is, therefore, quite surprising, as described in more detail hereinafter, that restoration of a wild-type rph gene in E. coli K-12 strains containing the frameshift mutation can increase the amount of heterologous protein produced in such strains and, furthermore, facilitate the improved purification and yield of heterologous proteins manufactured in these host cells.